EndoS from Streptococcus pyogenes is hydrolyzed by the cysteine proteinase SpeB and requires glutamic acid 235 and tryptophans for IgG glycan-hydrolyzing activity

Abstract

Background: The endoglycosidase EndoS and the cysteine proteinase SpeB from the human pathogen Streptococcus pyogenes are functionally related in that they both hydrolyze IgG leading to impairment of opsonizing antibodies and thus enhance bacterial survival in human blood. In this study, we further investigated the relationship between EndoS and SpeB by examining their in vitro temporal production and stability and activity of EndoS. Furthermore, theoretical structure modeling of EndoS combined with site-directed mutagenesis and chemical blocking of amino acids was used to identify amino acids required for the IgG glycan-hydrolyzing activity of EndoS.

Results: We could show that during growth in vitro S. pyogenes secretes the IgG glycan-hydrolyzing endoglycosidase EndoS prior to the cysteine proteinase SpeB. Upon maturation SpeB hydrolyzes EndoS that then loses its IgG glycan-hydrolyzing activity. Sequence analysis and structural homology modeling of EndoS provided a basis for further analysis of the prerequisites for IgG glycan-hydrolysis. Site-directed mutagenesis and chemical modification of amino acids revealed that glutamic acid 235 is an essential catalytic residue, and that tryptophan residues, but not the abundant lysine or the single cysteine residues, are important for EndoS activity.

Conclusion: We present novel information about the amino acid requirements for IgG glycan-hydrolyzing activity of the immunomodulating enzyme EndoS. Furthermore, we show that the cysteine proteinase SpeB processes/degrades EndoS and thus emphasize the importance of the SpeB as a degrading/processing enzyme of proteins from the bacterium itself.

Figure 1

Secretion of EndoS and SpeB during growth of S. pyogenes strain AP1. Panel A, growth of wild type AP1 and measurements of optical density at 620 nm over time. Samples were withdrawn for analysis at indicated time points. Panel B, detection of EndoS in samples from indicated time points using antiserum against full-length EndoS. Panel C, detection of SpeB in the culture supernatants using antiserum against the 40 kDa zymogen form of SpeB.

Figure 2

EndoS is hydrolyzed by SpeB. Panel A, analysis of SpeB-hydrolysis of EndoS during growth of the wild type AP1 (lanes A and B), the SpeB mutant AL1 (lane C and D), and the EndoS mutant MC14 (lanes E and F) with antiserum against EndoS and SpeB. Presence or absence of DTT during growth is indicated under the lower panel. Arrows to the right indicate the positions of full-length EndoS (FL), the zymogen (Z) and proteinase (P) forms of SpeB. Panel B, rEndoS incubated with increasing amounts of SpeB (lanes A-E) or thermolysin (lane F) as indicated and analyzed by SDS-PAGE. Arrows to the right indicate the positions of the 46- and 62-kDa forms of EndoS, SpeB, and thermolysin. Panel C, densitometric analysis of whole lanes A-E (excluding SpeB) in panel B. Values are presented as area under the curve in pixels.

Figure 3

Possible domain organization and sequence alignments of EndoS. Panel A, schematic representation of the 995 amino acids EndoS. Ss indicates signal peptide, the chitinase family 18 active site motif in the amino-terminal domain is indicated, the SpeB cleavage site is indicated with an arrow, and the putative leucine-rich repeat region (LRR) is shown. Panel B, ClustalW alignment of EndoS and leucine-rich proteins from P. gingivalis (NP_905954), C. tetani (NP_781184), and L. monocytogenes (NP_463795). Amino acid number one corresponds to amino acid 446 in the whole protein. Panel C, RADAR repeat analysis showing three 37 amino acids leucine rich repeats and two additional repeats. Numbering is based on the 995 amino acids sequence of full-length EndoS.

Figure 4

Homology modeling of EndoS. Panel A, model of amino acids 37–446 of EndoSusing EndoF₃ as the template. Glu-235 in the active is shown in red. Panel B, EndoS model (yellow) superimposed in the structure of EndoF₃ (blue). Panel C, model of amino acids 446–557 of EndoS using the LRR-region from InlB as the template. Panel A and C, β-strands are shown in yellow, α-helices in purple, and loops in turquoise.

Figure 5

Glutamic acid 235 and tryptophan residues are important for EndoS activity. Panel A, homology model of the amino-terminal part of EndoS with glutamic acid 235 (red) and the tryptophan residues (blue) are highlighted. Panel B, dilutions of rEndoS or rEndoS(E235Q) incubated with 1 μg human IgG for 1 hour at 37°C. IgG-glycan hydrolysis by EndoS was analyzed by SDS-PAGE and LCA-blot. Panel C, human IgG incubated with buffer, rEndoS modified with NEM, NBS, IAA, or unmodified (Buffer). The proteins were separated by SDS-PAGE, stained or analyzed for LCA reactivity in blot.

Figure 6

SpeB-hydrolyzed rEndoS loses activity on human IgG. Panel A, rEndoS was incubated with SpeB as described in material and methods at indicated time points and 10 μl of samples were applied on polyacrylamide gel and stained with Coomassie Blue. Panel B, 2 μl sample withdrawn from the same incubations, was incubated with 2 g IgG for 2 hours and IgG hydrolysis was analyzed by SDS-PAGE and LCA-blot.